1. Field of the Invention
This invention is directed to a method for screening patients suspected of having Alzheimer's disease. In particular, this invention involves a diagnostic procedure in which the amount of .beta.-amyloid precursor protein (APP) retained on the plasma membrane of activated platelets from the patient is compared to the amount retained by control platelets.
2. Review of Related Art
Selkoe (1994), Ann. Rev. Neurosci., 17:489-517, and J. Neuropath. Exp. Neurol., 53:438-447, report that amyloid .beta.-peptide (A.beta.) appears in many different forms in anatomically diverse locations in Alzheimer's patients, as well as Downs' Syndrome patients and even to some extent in normally aging individuals. A.beta. has been identified as a small fragment of a large protein called the amyloid precursor protein (APP) which is produced as a transmembrane protein; the A.beta. peptide sequence is found within APP in a position overlapping the APP transmembrane domain. Both A.beta. and the extracellular domain of APP have been observed to be secreted, but the levels of secreted peptide have not been shown to be diagnostically correlated with the presence or absence of Alzheimer's disease. Cleavage of APP can occur by more than one route, and the route can be monitored immunologically, by determining whether particular epitopes on intact or soluble forms of APP and/or A.beta. can be detected.
Bush, et al. (1992), Ann. Neurol., 32:57-65, pointed out that a blood test correlated with Alzheimer's disease would be particularly useful. To that end, diverse soluble fragments of APP were characterized, and it is reported that large fragments (130 kDa N-terminal fragment of APP) were present in Alzheimer's patients blood at higher concentration than in normal controls.
It has been well documented that human platelet .alpha.-granules contain large amounts (&gt;95%) of the circulating APP. Furthermore, there has been some evidence that, even in resting platelets, some of the APP may already have been proteolytically processed. Upon activation by physiological stimuli such as thrombin, platelets release their granules' contents, secreting any soluble fragments into the exterior medium, and translocating any granule membrane proteins to the plasma membrane so that the portion previously facing into the interior of the granule now faces out into the medium.
Schlossmacher, et al. (1992), Neurobiol. Aging, 13:421-434, reported that full length APP is retained on the membranes of activated platelets, and fragments missing amyloid .beta.-peptide (A.beta.) are released. The studies were performed on normal platelets, and no attempt was made to compare these to platelets from Alzheimer's patients. Li, et al. (1995), J. Biol. Chem., 270:14140-14147, also reported on APP processing by normal platelets. APP was reported to be cleaved to form a large soluble fragment and a small membrane-bound fragment containing the .beta.-amyloid protein (A.beta.) before activation of platelets. Upon activation, these two fragments of APP were reported to be further degraded in normal platelets.
Smith, et al. (1992), Blood, 80:2252-2260, reported that platelets secrete fragments of APP when activated. It was also reported that thrombin specifically cleaves soluble APP, and it was suggested that similar cleavage in vivo would leave small membrane-bound fragments containing the amyloid .beta.-peptide (A.beta.) on platelet membranes. In contrast, Bush, et al. (1993), Ann. N.Y. Acad. Sci., 695:175-182, investigated APP metabolism in various blood cells and reported that APP is present in amyloidogenic form in circulating platelets, which have been suggested as models for study of neuronal diseases. However, this study found no correlation between any variations in APP metabolism in platelets and Alzheimer's disease. Li, et al. (1994), Blood, 84:133-142, also reported no difference in the total amount of platelet membrane-associated APP found between platelets from Alzheimer's disease patients and controls.
Davies, et al. (1993), Biochem. Biophys. Res. Comm., 194:537-543, described platelet responses to thrombin stimulation as: (a) increase in cytoplasmic calcium ion; (b) membrane depolarization; (c) transient acidification followed by alkalinization of the cytoplasm; and (d) degranulation, including sequential release of dense granule contents, .alpha.-granule contents (including APP), and lysosomal granule contents. The extent of acidification in platelets of probable Alzheimer's disease patients was reported to exceed that of control platelets, but no mechanism for this effect or consequential effects on proteolytic processing of APP was described.
Despite extensive study of the fate of APP in various cells of Alzheimer's patients and normal controls, including platelets, the need for a blood test correlated with Alzheimer's disease pointed out by Bush, et al. (1992) is still unmet.